scholarly journals Evaluation of Level of Agreement in Bordetella Species Identification in Three U.S. Laboratories during a Period of Increased Pertussis

2015 ◽  
Vol 53 (6) ◽  
pp. 1842-1847 ◽  
Author(s):  
Brunilís Burgos-Rivera ◽  
Adria D. Lee ◽  
Katherine E. Bowden ◽  
Amanda E. Faulkner ◽  
Brent L. Seaton ◽  
...  
Keyword(s):  
Species Identification ◽  
Insertion Sequence ◽  
The United States ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Content Type ◽  
Negative Results ◽  
Relative Contribution ◽  
Level Of Agreement ◽  
Good Agreement

While PCR is the most common method used for detectingBordetella pertussisin the United States, most laboratories use insertion sequence481(IS481), which is not specific forB. pertussis; therefore, the relative contribution of otherBordetellaspecies is not understood. The objectives of this study were to evaluate the proportion of otherBordetellaspecies misidentified asB. pertussisduring a period of increased pertussis incidence, determine the level of agreement inBordetellaspecies detection between U.S. commercial laboratories and the CDC, and assess the relative diagnostic sensitivity of CDC's PCR assay when using a different PCR master mix. Specimens collected between May 2012 and May 2013 were tested at two U.S. commercial laboratories forB. pertussisandB. parapertussisdetection. Every fifth specimen positive for IS481and/or IS1001with cycle threshold (CT) values of ≤35 was sent to CDC for PCR testing that identifiesBordetellaspecies. Specimens with indeterminate or negative results in the CDC PCR were tested using an alternate PCR master mix. Of 755 specimens, there was agreement in species identification for 83.4% (n= 630). Of the specimens with different identifications (n= 125), 79.2% (n= 99) were identified as indeterminateB. pertussisat CDC. Overall, 0.66% (n= 5) of the specimens were identified asB. holmesiiorB. bronchisepticaat CDC. Of 115 specimens with indeterminate or negative results, 46.1% (n= 53) wereB. pertussispositive when tested by an alternate master mix, suggesting a possible increase in assay sensitivity. This study demonstrates good agreement between the two U.S. commercial laboratories and CDC and little misidentification ofBordetellaspecies during the 2012 U.S. epidemic.

scholarly journals Multicenter Clinical Evaluation of the Automated Aries Group A Strep PCR Assay from Throat Swabs

2018 ◽  
Vol 57 (2) ◽  
Author(s):  
N. Kanwar ◽  
J. Crawford ◽  
C. Ulen ◽  
T. S. Uphoff ◽  
J. Dien Bard ◽  
...  
Keyword(s):  
Predictive Value ◽  
Throat Swab ◽  
False Negative ◽  
Reference Method ◽  
The United States ◽  
Antibiotic Administration ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Content Type ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) is one of the leading causes of bacterial pharyngitis. Early GAS diagnosis is critical for appropriate antibiotic administration that reduces the risk of GAS sequelae and limits spread of the infection. The Aries Group A Strep (GAS) assay (Luminex, Austin, TX) is a fully automated PCR assay for direct detection of GAS in throat swab specimens in less than 2 h with minimum hands-on time. This multicenter prospective study evaluated the clinical performance of the Aries GAS assay compared to that of Streptococcus pyogenes culture. Subjects with symptoms consistent with pharyngitis were enrolled across four sites in the United States, and a throat swab in liquid Amies medium was obtained. Aries and reference testing was performed within 72 and 48 h after sample collection, respectively. Of 623 throat swab specimens from patients with pharyngitis (93.6% <18 years old, 54.3% female), the reference method yielded valid results for 618 specimens. Reference and Aries assay testing showed GAS-positive results for 160 (25.9%) and 166 (26.9%) specimens, respectively. Compared to the reference method, Aries assay sensitivity was 97.5% (95% confidence interval [CI], 93.7% to 99.0%), specificity was 97.8% (95% CI, 96.0 to 98.8%), positive predictive value was 94.0% (95% CI, 89.3% to 96.7%), and negative predictive value was 99.1% (95% CI, 97.7% to 99.7%). There were 10 false-positive and four false-negative detections with the Aries assay. Discrepant analysis with bidirectional sequencing yielded concordant results with the Aries assay for nine of 14 discordant samples. The Aries assay had high sensitivity and specificity for qualitative detection of group A Streptococcus from patients with pharyngitis.

scholarly journals Performance and Verification of a Real-Time PCR Assay Targeting thegyrAGene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae: TABLE 1

2016 ◽  
Vol 54 (3) ◽  
pp. 805-808 ◽  
Author(s):  
P. Hemarajata ◽  
S. Yang ◽  
O. O. Soge ◽  
R. M. Humphries ◽  
J. D. Klausner
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
The United States ◽  
Test Results ◽  
Pcr Assay ◽  
Content Type ◽  
Agar Dilution ◽  
Ciprofloxacin Resistance ◽  
To Receive

In the United States, 19.2% ofNeisseria gonorrhoeaeisolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive forN. gonorrhoeaeby the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

scholarly journals Comparison of Multilocus Sequence Typing and the Xpert C. difficile/Epi Assay for Identification of Clostridium difficile 027/NAP1/BI

2015 ◽  
Vol 54 (3) ◽  
pp. 775-778 ◽  
Author(s):  
Tracy McMillen ◽  
Mini Kamboj ◽  
N. Esther Babady
Keyword(s):  
United States ◽  
Clostridium Difficile ◽  
Confidence Interval ◽  
Multilocus Sequence Typing ◽  
The United States ◽  
Content Type ◽  
Presumptive Identification ◽  
Good Agreement

Clostridium difficile027/NAP1/BI is the most commonC. difficilestrain in the United States. The XpertC. difficile/Epi assay allows rapid, presumptive identification ofC. difficileNAP1. We compared XpertC. difficile/Epi to multilocus sequence typing for identification ofC. difficileNAP1 and found “very good” agreement at 97.9% (κ = 0.86; 95% confidence interval, 0.80 to 0.91).

scholarly journals Mycobacterium arupense, Mycobacterium heraklionense, and a Newly Proposed Species, “Mycobacterium virginiense” sp. nov., but Not Mycobacterium nonchromogenicum, as Species of the Mycobacterium terrae Complex Causing Tenosynovitis and Osteomyelitis

2016 ◽  
Vol 54 (5) ◽  
pp. 1340-1351 ◽  
Author(s):  
Ravikiran Vasireddy ◽  
Sruthi Vasireddy ◽  
Barbara A. Brown-Elliott ◽  
Nancy L. Wengenack ◽  
Uzoamaka A. Eke ◽  
...  
Keyword(s):  
United States ◽  
16S Rrna ◽  
Species Identification ◽  
Type Strain ◽  
Molecular Taxonomy ◽  
The United States ◽  
Undescribed Species ◽  
Content Type ◽  
Sequence Identity ◽  
As Species

Mycobacterium terraecomplex has been recognized as a cause of tenosynovitis, withM. terraeandMycobacterium nonchromogenicumreported as the primary etiologic pathogens. The molecular taxonomy of theM. terraecomplex causing tenosynovitis has not been established despite approximately 50 previously reported cases. We evaluated 26 isolates of theM. terraecomplex associated with tenosynovitis or osteomyelitis recovered between 1984 and 2014 from 13 states, including 5 isolates reported in 1991 asM. nonchromogenicumby nonmolecular methods. The isolates belonged to three validated species, one new proposed species, and two novel related strains. The majority of isolates (20/26, or 77%) belonged to two recently described species:Mycobacterium arupense(10 isolates, or 38%) andMycobacterium heraklionense(10 isolates, or 38%). Three isolates (12%) had 100% sequence identity to each other by 16S rRNA and 99.3 to 100% identity byrpoBgene region V sequencing and represent a previously undescribed species within theM. terraecomplex. There were no isolates ofM. terraeorM. nonchromogenicum, including among the five isolates reported in 1991. The 26 isolates were susceptible to clarithromycin (100%), rifabutin (100%), ethambutol (92%), and sulfamethoxazole or trimethoprim-sulfamethoxazole (70%). The current study suggests thatM. arupense,M. heraklionense, and a newly proposed species (“M. virginiense” sp. nov.; proposed type strain MO-233 [DSM 100883, CIP 110918]) within theM. terraecomplex are the major causes of tenosynovitis and osteomyelitis in the United States, with little change over 20 years. Species identification within this complex requires sequencing methods.

scholarly journals Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica

2016 ◽  
Vol 54 (11) ◽  
pp. 2681-2688 ◽  
Author(s):  
S. Madison-Antenucci ◽  
R. F. Relich ◽  
L. Doyle ◽  
N. Espina ◽  
D. Fuller ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Cryptosporidium Parvum ◽  
Fluorescent Antibody ◽  
Giardia Duodenalis ◽  
The United States ◽  
Parasitic Infections ◽  
Pcr Assay ◽  
Cryptosporidium Hominis ◽  
Content Type ◽  
Common Causes

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis , Entamoeba histolytica , Cryptosporidium parvum , and Cryptosporidium hominis . Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA ( G. duodenalis and C. parvum or C. hominis ) or trichrome stain ( E. histolytica ). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis , 95.5% and 99.6 for C. parvum or C. hominis , and 100% and 100% for E. histolytica , respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

scholarly journals Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (8) ◽  
pp. 2589-2595 ◽  
Author(s):  
Feifei Han ◽  
Fei Wang ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Vibrio Vulnificus ◽  
Lamp Assay ◽  
The United States ◽  
Isothermal Amplification ◽  
Quantitative Detection ◽  
Strain Atcc ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results

ABSTRACTVibrio vulnificusis a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmentalV. vulnificusstrains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study,vcgCwas selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulentV. vulnificusstrains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulentV. vulnificusstrain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 103CFU/g ofV. vulnificusATCC 33815, while showing negative results for a nonvirulentV. vulnificusstrain (515-4c2) spiked at 107CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulentV. vulnificusstrain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulentV. vulnificusstrain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulentV. vulnificusin raw oysters with high speed, specificity, and sensitivity, which may facilitate better control ofV. vulnificusrisks associated with raw oyster consumption.

scholarly journals Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood

2015 ◽  
Vol 53 (7) ◽  
pp. 2251-2257 ◽  
Author(s):  
Martina I. Lefterova ◽  
Indre Budvytiene ◽  
Johanna Sandlund ◽  
Anna Färnert ◽  
Niaz Banaei
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Whole Blood ◽  
18S Rrna ◽  
Pcr Assay ◽  
Test Species ◽  
Content Type ◽  
The Real ◽  
Two Samples

Malaria is the leading identifiable cause of fever in returning travelers. AccuratePlasmodiumspecies identification has therapy implications forP. vivaxandP. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay forPlasmodiumspecies identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% forP. falciparum(20/21 positives detected) and 100% for thePlasmodiumgenus (52/52),P. vivax(20/20),P. ovale(9/9), andP. malariae(6/6). The sensitivity of theP. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% forP. vivax(49/52) and 100% forP. falciparum(51/51),P. ovale(62/62),P. malariae(69/69), andP. knowlesi(52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixedP. falciparumandP. ovaleinfection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitivePlasmodiumspecies identification shortly after malaria diagnosis by microscopy.

scholarly journals Multiplex PCR Analysis for Rapid Detection of Klebsiella pneumoniae Carbapenem-Resistant (Sequence Type 258 [ST258] and ST11) and Hypervirulent (ST23, ST65, ST86, and ST375) Strains

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Fangyou Yu ◽  
Jingnan Lv ◽  
Siqiang Niu ◽  
Hong Du ◽  
Yi-Wei Tang ◽  
...  
Keyword(s):  
United States ◽  
Klebsiella Pneumoniae ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
The United States ◽  
Sequence Type ◽  
Pcr Assay ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Resistant Strains

ABSTRACT Carbapenem-resistant and hypervirulent Klebsiella pneumoniae strains have emerged recently. These strains are both hypervirulent and multidrug resistant and may also be highly transmissible and able to cause severe infections in both the hospital and the community. Clinical and public health needs require a rapid and comprehensive molecular detection assay to identify and track the spread of these strains and provide timely infection control information. Here, we develop a rapid multiplex PCR assay capable of distinguishing K. pneumoniae carbapenem-resistant isolates of sequence type 258 (ST258) and ST11, and hypervirulent ST23, ST65/ST375, and ST86 clones, as well as capsular types K1, K2, K locus type 47 (KL47), and KL64, and virulence genes rmpA, rmpA2, iutA, and iroN. The assay demonstrated 100% concordance with 118 previously genotyped K. pneumoniae isolates and revealed different populations of carbapenem-resistant and hypervirulent strains in two collections in China and the United States. The results showed that carbapenem-resistant and hypervirulent K. pneumoniae strains are still rare in the United States, whereas in China, ∼50% of carbapenem-resistant strains carry rmpA/rmpA2 and iutA virulence genes, which are largely associated with the epidemic ST11 strains. Similarly, a high prevalence of hypervirulent strains was found in carbapenem-susceptible isolates in two Chinese hospitals, but these primarily belong to ST23, ST65/ST375, and ST86, which are distinct from the carbapenem-resistant strains. Taken together, our results demonstrated that this PCR assay can be a useful tool for molecular surveillance of carbapenem-resistant and hypervirulent K. pneumoniae strains.

scholarly journals Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

2015 ◽  
Vol 59 (9) ◽  
pp. 5574-5580 ◽  
Author(s):  
Peera Hemarajata ◽  
Shangxin Yang ◽  
Janet A. Hindler ◽  
Romney M. Humphries
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Hydrolytic Activity ◽  
The United States ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Content Type ◽  
High Resolution Melt Analysis ◽  
Carbapenem Resistant

ABSTRACTThe rapid global spread of carbapenem-resistantEnterobacteriaceae(CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, usingblaOXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, includingblaKPC,blaSME,blaIMP,blaNDM-1,blaVIM,blaOXA-48,blaOXA-162,blaOXA-181,blaOXA-204,blaOXA-244,blaOXA-245, andblaOXA-232. Our assay identified the presence ofblaOXA-48-likeβ-lactamase genes and clearly distinguished betweenblaOXA-48and its variants in control strains, including betweenblaOXA-181andblaOXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.

scholarly journals Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance

2015 ◽  
Vol 53 (8) ◽  
pp. 2706-2708 ◽  
Author(s):  
Cameron Buckley ◽  
Ella Trembizki ◽  
Robert W. Baird ◽  
Marcus Chen ◽  
Basil Donovan ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Direct Detection ◽  
False Negative ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Negative Results ◽  
False Negative Results ◽  
Sequence Variations

A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.

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